Norwalk virus (NV) is classified as a human calicivirus (family Caliciviridae, genus Norovirus) and is the major cause of acute gastroenteritis in humans. (Jiang et al., “Norwalk Virus Genome Cloning and Characterization,” Science 250:1580-1583 (1990), Clarke et al., “Organization and Expression of Calicivirus Genes,” J Infect Dis 181(Suppl 2):S309-316 (2000)). “Norovirus” was recently approved as the official genus name for the group of viruses provisionally described as “Norwalk-like viruses” (NLV). This group of viruses has also been referred to as caliciviruses (because of their virus family name) and as small round structured viruses, or SRSVs (because of their morphologic features).
Caliciviruses (family Caliciviridae) infect animals and humans. Within the family Caliciviridae, four genera have been distinguished: Vesivirus Lagovirus, Norovirus, and Sapovirus (until recently known as Sapporo-like viruses (SLV)(van der Poel et al., “Norwalk-Like Calicivirus Genes in Farm Animals,” Emerging Infectious Diseases 6(1):36-41 (2000); Pringle, C. R., “Virus Taxonomy” Arch Virol 143:1449-1459 (1998)). The genera Vesivirus and Lagovirus contain a broad range of animal caliciviruses, but viruses in the NLV and SLV genera until recently had been found only in humans. Viruses closely related to Norwalk-like viruses have now been found in calves and pigs (van der Poel et al., “Norwalk-Like Calicivirus Genes in Farm Animals,” Emerging Infectious Diseases 6(1):36-41 (2000)), and a murine Norwalk-like virus has been identified (Karst et al., “STAT-1-Dependent Innate Immunity to a Norwalk-Like Virus” Science 299:1575-1578 (2003)).
Noroviruses are named after the original strain “Norwalk virus,” which caused an outbreak of gastroenteritis in a school in Norwalk, Ohio, in 1968. Currently, there are at least four Norovirus genogroups (GI, GII, GIII, and GIV), which in turn are divided into approximately 20 genetic clusters. The caliciviruses are grouped on the basis of morphology, size, protein profile, and nucleic acid. Norwalk virus and some other human caliciviruses share considerable genetic homology.
Recent estimates indicate that NV and NV-like agents are responsible for greater than 90% of outbreaks of acute nonbacterial gastroenteritis in developed and developing countries (Fankhauser et al., “Molecular Epidemiology of “Norwalk-Like Viruses” in Outbreaks of Gastroenteritis in the United States,” J Infect Dis 178:1571-1578 (1998), Vinje et al., “The Incidence and Genetic Variability of Small Round-Structured Viruses in Outbreaks of Gastroenteritis in The Netherlands,” J Infect Dis 176:1374-1378 (1997)). Centers for Disease Control in Atlanta attributes about 181,000 cases of gastrointestinal illness in the U.S. each year to NV. Viral gasteroenteritis affects so many individuals that only the common cold is reported more frequently than viral gastroenteritis. Viral transmission is generally by the fecal-oral route. Symptoms of the disease include nausea, vomiting, acute diarrhea, and stomach cramps, with infection most common in adults or older children. The virus has a one to two day incubation period, with a two to three day recovery; however, individuals may still be highly infectious after recovery. Infection with Norwalk virus confers some immunity, which declines over 24 months, allowing re-infection. Epidemic outbreaks of this disease frequently occur on cruise ships, in schools, day care centers, nursing homes, hospitals, and communities. The increasing clinical significance of these infections suggests the pressing need for an efficacious vaccine against Norwalk virus (Estes et al., “Norwalk Virus Vaccines: Challenges and Progress,” J Infect Dis 181(Suppl 2):S367-373 (2000)). However, to date, it is not cultivatable in vitro, and there are no animal models for study or culturing the virus. The lack of Norwalk virus animal models is apparently related to the fact that humans are the only known host for Norwalk virus.
Norwalk virus is a round, nonenveloped, 27-nm virion. Its nucleic acid contains single-stranded, positive-sense RNA. It has a single structural protein characteristic of a calicivirus. The single, positive strand of Norwalk virus RNA contains three open reading frames, the second of which is known to encode a single NV capsid protein (NVCP) that self-assembles into empty virus-like particles (VLPs) lacking viral RNA when expressed in the baculovirus/insect cell expression system (Jiang et al., “Expression, Self-Assembly, and Antigenicity of the Norwalk Virus Capsid Protein,” J Virol 66:6527-6532 (1992), Jiang et al., “Sequence and Genomic Organization of Norwalk Virus,” Virology 195:51-61 (1993)) and plant cells (Mason et al., “Expression of Norwalk Virus Capsid Protein in Transgenic Tobacco and Potato and its Oral Immunogenicity in Mice,” Proc Natl Acad Sci USA 93:5335-5340 (1996)). X-ray crystallography of recombinant NV VLPs (rNV VLPs) showed that these VLPs are composed of 90 dimers of the NVCP that form T=3 icosahedral structure with a diameter of about 38 nm (Prasad et al., “X-ray Crystallographic Structure of the Norwalk Virus Capsid,” Science 286:287-290 (1999)). The rNV VLPs are stable at low pH, when lyophilized, and when stored long term at 4° C. (Estes et al., “Norwalk Virus Vaccines: Challenges and Progress,” J Infect Dis 181(Suppl 2):S367-373 (2000)). The insect cell-derived VLPs are immunogenic in experimental animals and in human volunteers following oral administration (Ball et al., “Oral Immunization with Recombinant Norwalk Virus-Like Particles Induces a Systemic and Mucosal Immune Response in Mice,” J Virol 72:1345-1353 (1998), Ball et al., “Recombinant Norwalk Virus-Like Particles Given Orally to Volunteers: Phase I Study,” Gastroenterology 117:40-48 (1999)), and in mice when administered parenterally (Jiang et al., “Expression, Self-Assembly, and Antigenicity of the Norwalk Virus Capsid Protein,” J Virol 66:6527-6532 (1992)), and intranasally (Guerrero et al., “Recombinant Norwalk Virus-Like Particles Administered Intranasally to Mice Induce Systemic and Mucosal (Fecal and Vaginal) Immune Responses.,” J Virol 75:9713-9722 (2001)). These qualities make the rNV VLPs useful as a candidate for vaccine against Noroviruses, including Norwalk virus.
Thus, what is needed now is a strategy which utilizes what has been elucidated about the genetic structure of Noroviruses to produce a superior, cost-efficient, effective, and stable vaccine against Noroviruses, including Norwalk virus, and a method to produce such a vaccine on a large-scale for distribution and human use world-wide.
The present invention is directed to overcoming these and other deficiencies in the art.